Apparatus and method of using a simulated skin substrate for testing insect repellants

ABSTRACT

An apparatus includes a housing having an aperture extending through a wall of the housing, a carbon dioxide delivery device coupled to the housing through the aperture, a non-biological skin substitute substrate, and a heater coupled to the substrate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/650,516, filed Jul. 14, 2017, which claims priority to U.S.Provisional Patent Application No. 62/363,061, filed on Jul. 15, 2016,each of which is incorporated herein by reference as if set forth in itsentirety.

REFERENCE REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable

SEQUENCE LISTING

Not applicable

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure generally relates to an apparatus for evaluatingthe effectiveness of insect repellents on human skin using a simulatedskin substrate and methods for using same. The method is particularlyuseful and suitable for testing the efficacy of skin-applied insectrepellents and the attraction of biting insects to human subjectswithout using human testing participants.

2. Description of the Background of the Invention

Insect-transmitted diseases have long been prevalent, however there aresignificant hurdles to the development of new effective insectrepellents because of the difficulties of testing the efficacy of thosenew repellents. Particularly, the testing of skin-applied insectrepellents and the attraction of biting insects to human subjects isdifficult to test without using human test subjects.

Efficacy tests without using human subjects have many benefits. Ingeneral, tests involving human subjects tend to have higher operationalcosts and complicated testing protocols. When human subjects are notemployed in tests, experimental costs can be reduced significantly.Further, tests using human surrogate apparatuses can be conductedwithout geographic or environmental limitations. Particularly, similartests can be conducted in multiple environments, e.g., a temperate ortropical zone, to reduce the number of variables except for those uniqueto each geographic locale. All of these benefits assist in makingimproved repellents based on the location of the consumer.

A need exists for human surrogate testing apparatuses and methods fordetermining the effectiveness of insect repellents. Several previouslydeveloped tests involve using a collagen membrane or a piece of fabricover a warmed feeding chamber containing blood, artificial blood, orwarm water containing stimulants for insects. However, these tests andmethods do not provide comparable testing results to those derived fromtests involving human subjects, and collagen-based substrates do notpermit repeated testing cycles. Therefore, it is important to develop ahuman surrogate apparatus and method for testing the effectiveness ofinsect repellents using a non-collagen based substrate, which providesidentical or substantially similar results to tests involving humanparticipants without having the limitations associated with collagensubstrates.

SUMMARY OF THE INVENTION

The present disclosure presents methods and apparatuses for testing theefficacy of skin-applied repellents by employing a simulated humannon-collagen based skin substrate. In certain embodiments, a method fortesting the efficacy of skin-applied insect repellents comprises thesteps of providing a skin substitute substrate, exposing biting insectsto the skin substitute substrate treated with an insect repellent,blocking the treated skin substitute substrate from the plurality ofinsects for a second period of time, repeating the exposing step and theblocking step for (n) times until at least one insect lands on thetreated skin substitute substrate for greater than a third period oftime during the (n) exposure step and during a subsequent (n+1) exposurestep, and recording a complete protection time (“CPT”).

In certain embodiments a method for testing the efficacy of askin-applied insect repellent comprises the steps of providing anon-biological skin substitute substrate simulating at least onephysical, chemical, or biological characteristic of human skin, treatingthe skin substitute substrate with an insect repellent, exposing aplurality of insects to the treated skin substitute substrate for afirst period of time, blocking the treated skin substitute substratefrom the plurality of insects for a second period of time, repeating theexposing step and the blocking step for (n) times until at least oneinsect lands on the treated skin substitute substrate for greater than athird period of time during the (n)th exposure step and during asubsequent (n+1)th exposure step, and recording a fourth period of timeneeded to reach the (n)th exposure. In certain embodiments the methodcomprises the step of enclosing the treated skin substitute substrateand the plurality of insects in a housing. In certain embodiments thenon-biological skin substitute substrate is synthetic.

In certain embodiments the non-biological skin substitute substrate hasan R_(a) value of about 0.01 to about 0.2 μm. In certain embodiments thenon-biological skin substitute substrate has specular reflectance peakat a wavelength of about 306 nm. In certain embodiments thenon-biological skin substitute substrate has a diffuse reflectanceexhibiting properties associated with a Lambertian reflectance. Incertain embodiments the contact angle between the insect repellent and asurface of the non-biological skin substitute substrate approaches or issubstantially zero.

In certain embodiments, the treated non-biological skin substitutesubstrate is exposed to biting insects for about 1 minute to about 5minutes and withdrawn from biting insects for about 25 minutes to about29 minutes. In one particular embodiment, the treated skin substitutesubstrate is exposed to biting insects for about 1 minute and withdrawnfrom biting insects for about 29 minutes. This cycle is repeated untilone or more biting insects land on the treated substrate for equal to orlonger than 2 seconds. If in the subsequent exposure, one or more bitinginsects land on the treated substrate again for equal to or longer than2 seconds, the repeated cycle is terminated and the last exposure timeis the CPT of the tested repellent. Collagen-based substrates cannot beused in assays comprising multiple testing cycles.

In some embodiments, additional steps of the method may includeproviding a housing comprising a plurality of insects, supplying thehousing with carbon dioxide (CO₂) at a rate of about 400 mL/min (14oz/min) to about 600 mL/min (20.3 oz/min), applying an amount ofskin-applied insect repellent to the skin substitute substrate to formthe treated substrate, and maintaining a temperature of the skinsubstitute substrate within a range of about 35° C. to about 39° C.Further, in some embodiments the additional steps of the method maycomprise the step of placing the housing with the plurality of insectsunder a testing condition for about 30 minutes to acclimate theplurality of insects to the testing condition.

In certain embodiments, a method for testing the efficacy ofskin-applied insect repellents comprises the steps of providing asynthetic, non-biological skin substitute substrate, treating thesynthetic, non-biological skin substitute substrate with an insectrepellent, exposing a plurality of insects to the treated skinsubstitute substrate for a first period of time, blocking the treatedskin substitute substrate from the plurality of insects for a secondperiod of time, repeating the exposing step and the blocking step for(n) times until at least one insect lands on the treated skin substitutesubstrate for greater than a third period of time during the (n)thexposure step and during a subsequent (n+1)th exposure step, andrecording a fourth period of time needed to reach the (n)th exposure,wherein the treated skin substitute substrate and the plurality ofinsects are enclosed in a housing.

Certain embodiments of the method for testing the efficacy ofskin-applied insect repellents comprises exposing the plurality ofinsects in the housing to an untreated skin substitute substrate forabout 60 seconds, recording the number of insects that land on theuntreated substrate for longer than about 2 seconds, and determiningwhether the plurality of insects in the housing need to be replaced,wherein the determination to replace occurs when the recorded number ofinsects that land is less than 20.

In other embodiments, a human surrogate apparatus for testing theefficacy of skin-applied repellents is disclosed. In certain embodimentsthe apparatus for testing the efficacy of a skin-applied insectrepellent comprises a housing comprising an aperture extending through awall of the housing, a carbon dioxide delivery device coupled to thehousing through the aperture, a non-biological skin substitutesubstrate, and a heater coupled to the non-biological skin substitutesubstrate. In certain embodiments the non-biological skin substitutesubstrate simulates at least one physical, chemical, or biologicalcharacteristic of human skin. In certain embodiments the non-biologicalskin substrate is synthetic. Further, when the housing is open, insectswithin the housing will have access to the skin substitute substrate;and when the housing is closed, the insects will not have access to theskin substitute substrate.

In certain embodiments, the non-biological skin substitute substrate hasan R_(a) value of about 0.01 to about 0.2 μm. In certain embodiments thenon-biological skin substitute substrate has specular reflectance peakat a wavelength of about 306 nm. In certain embodiments thenon-biological skin substitute substrate has a diffuse reflectanceexhibiting properties associated with a Lambertian reflectance. Incertain embodiments the contact angle between the insect repellent and asurface of the non-biological skin substitute substrate approaches or issubstantially zero.

In certain embodiments, a portable human surrogate apparatus for fieldtesting the efficacy of skin-applied repellents is disclosed. Anembodiment of the portable human surrogate apparatus comprises aportable housing with a cavity, a heater disposed within the cavity ofthe housing, a temperature buffering device coupled to the heater anddisposed within the cavity of the housing, a non-biological skinsubstitute substrate coupled to the temperature buffering device anddisposed within the cavity of the housing, and a carbon dioxide deliverydevice coupled to the housing.

In certain embodiments, a method for measuring mosquito landing pressureis disclosed. An embodiment of the method comprises providing anon-biological skin substitute substrate, exposing a plurality ofmosquitos to the skin substrate, and recording a number of mosquitoslanding on the skin substrate during a period of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of a human surrogate apparatus fortesting the effectiveness of insect repellents;

FIG. 2 demonstrates an embodiment of a field-portable human surrogateapparatus for testing the effectiveness of insect repellents;

FIG. 3 is a schematic cutaway view illustrating components of thefield-portable human surrogate apparatus of FIG. 2;

FIG. 4 is a flowchart illustrating the method of using the humansurrogate apparatus of FIGS. 1 and 2 to test the efficacy of an insectrepellent;

FIG. 5 is a scatter plot showing comparisons of complete protection time(“CPT”) of N,N-Diethyl-3-methylbenzamide (“DEET”) in hours for testsinvolving human participants and tests involving a human surrogateapparatus according to an embodiment of the present disclosure in alaboratory;

FIG. 6 is a bar graph showing comparisons of mean CPT in hours withdifferent concentrations of DEET for tests involving human participantsand tests involving a human surrogate apparatus according to anembodiment of the present disclosure;

FIG. 7 is a graph comparing a selected mathematical model for CPT fortests involving human participants and tests involving a human surrogateapparatus according to an embodiment of the present disclosure;

FIG. 8 is a scatter plot showing comparisons of CPT in hours for 7% DEETbetween laboratory and field tests using a human surrogate apparatusaccording to an embodiment of the present disclosure;

FIGS. 9A and 9B illustrate the specular reflectance and diffusereflectance of the synthetic (non-biological) skin substitute substrate,namely, in this example, Vitro-Skin®;

FIG. 10A is a scatter plot showing comparisons of CPT in hours of DEETin field tests involving human participants and a human surrogateapparatus according to an embodiment of the present disclosure;

FIG. 10B is a graph comparing a selected mathematical model for CPT fortests conducted in FIG. 10A;

FIG. 10C shows comparisons of mean CPT in hours from FIG. 10A;

FIG. 11A is a scatter plot showing CPT in hours of2-(2-hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl ester(picaridin) for field tests involving human participants and a humansurrogate apparatus according to an embodiment of the presentdisclosure;

FIG. 11B is a graph comparing a selected mathematical model for CPT fortests conducted in FIG. 11A;

FIG. 11C shows comparisons of mean CPT in hours from FIG. 11A;

FIG. 12A is a scatter plot showing CPT in hours ofpara-menthane-3,8-diol (PMD) for field tests involving humanparticipants and a human surrogate apparatus according to an embodimentof the present disclosure;

FIG. 12B is a graph comparing a selected mathematical model for CPT fortests conducted in FIG. 12A;

FIG. 12C shows comparisons of mean CPT in hours from FIG. 12A;

FIG. 13 shows numbers of mosquitoes that landed on the skin substitutesubstrate when using a human surrogate apparatus according to anembodiment of the present disclosure and numbers of mosquitoes trappedby industry-standard mosquito traps in the field;

FIG. 14 illustrates a scatter plot showing comparisons of CPT ofpicaridin in hours for tests involving human participants and testsinvolving a human surrogate apparatus according to an embodiment of thepresent disclosure in a laboratory; and

FIG. 15 illustrates a scatter plot showing comparisons of CPT of PMD inhours for tests involving human participants and tests involving a humansurrogate apparatus according to an embodiment of the present disclosurein a laboratory.

Other aspects and advantages of the present invention will becomeapparent upon consideration of the following detailed description,wherein similar structures have similar reference numerals.

DETAILED DESCRIPTION

Embodiments of the current technology disclose human surrogateapparatuses and methods for using human surrogate apparatuses forconducting insect repellent evaluation tests. The disclosed humansurrogate apparatuses and methods provide identical or substantiallysimilar results to tests involving human participants for testing theeffectiveness of an insect repellent without the limitations associatedwith collagen-based simulated skin substrates. Further, the humansurrogate apparatuses according to an embodiment of the presentdisclosure are able to capture more mosquitoes compared toindustry-standard mosquito traps.

FIG. 1 illustrates one embodiment of a human surrogate apparatus 100 fortesting the efficacy of a skin-applied insect repellent. The humansurrogate apparatus 100 generally comprises a housing 102 for housinginsects, a carbon dioxide delivery device 104, a non-collagen based,non-biological substrate 106, and a heater 108. The non-collagen based,non-biological substrate 106 may be synthetic.

In an embodiment, housing 102 is a framed and cuboid shaped cage with astorage volume defined by a width of about 18 inches, a depth of about12 inches, and a height of about 12 inches. As described herein, “about”is used to mean a difference of plus or minus 10% in dimensionmeasurements. In this embodiment, the described dimensions define astorage volume of about 2592 cubic inches, which allows housing 102 tohouse at least 200 biting insects, such as lab-reared mosquitoes, suchas Aedes aegypti, during a testing. In other embodiments, the storagevolume may be at least 1000 cubic inches, or at least 2000 cubic inches,or at least 2500 cubic inches. In yet other embodiments, the storagevolume is sufficient to hold at least 100 mosquitoes, or at least 200mosquitoes, or at least 500 mosquitoes. Further, housing 102 maycomprise any shape insofar as it provides for a storage volumesufficient to hold a desired number of insects.

The construction of housing 102 may be varied based on numerous userconsiderations. In a preferred embodiment, housing 102 includes a topwall 110, a bottom wall 112, and side walls 114 a-d. In the embodimentdepicted in FIG. 1, the top wall 110 and the side walls 114 a-d comprisea polycarbonate material, or other clear plastic or glass material. Thebottom wall 112 comprises an opaque plastic or other material andincludes a sliding door 116. It is further contemplated that the slidingdoor 116 may be any type of door or resealable opening and positioned onone or more of the walls 110, 112, 114 a-d of housing 102. In thedepicted embodiment, the side walls 114 b and 114 c include a meshmaterial 118 extending across a portion thereof, providing for fluidcommunication between the internal storage volume and the externalatmosphere. More specifically, the mesh material may allow for airexchange and prevent CO₂ buildup in housing 102. Further, the side wall114 d includes a portion thereof comprising a fabric material 120, suchas, for example, a material found in a typical orthopedic tubularstockinet. The fabric material 120 can provide an opening through whichmosquitoes are placed in, or removed from, housing 102.

The substrate 106 is shown mounted to a disk 122, which is made ofmaterial that is able to transmit heat from a water tube 124 to thesubstrate 106. The disk 122 may comprise any shape, but preferablycomprises a shape that corresponds to that of the sliding door 116 andthat may be fittingly and/or sealingly received with and/or into anaperture defined by the sliding door. During testing, the disk 122 ispositioned next to the sliding door 116 with the substrate 106 disposedon a surface of the disk. Because of the complementary shaping andsizing of the sliding door 116 and the disk 122, the substrate 106 issealingly received within the housing 102 (or adjacent to the housing102) to prevent biting insects from traveling outside the container.

With reference still to the illustrated embodiment of FIG.1, the CO₂delivery device 104 delivers CO₂ through a tube 126 into the container102. The CO₂ delivery device 104 includes (or is in communication with)a pressurized source of CO₂, such as a pressurized CO₂ tank. In thepresent embodiment, the CO₂ is delivered at a constant rate of betweenabout 400 mL/min (14 oz/min) to about 600 mL/min (20.3 oz/min). In otherembodiments, the rate of CO₂ delivery may be provided in discretepulses, e.g., mimicking the temporal duration of human breathing. Otherranges of pulse or constant CO₂ flow rates are contemplated that providea periodic or constant flow of CO₂ within container 102 of between about500 to about 1500 parts per million (ppm) to stimulate the mosquitoesduring testing.

Moreover, in the depicted embodiment, heater 108 is a circulating waterheater and maintains the temperature of the substrate 106 during testingat about 35° C. to about 39° C. In alternative embodiments, othertemperatures are contemplated to simulate human conditions in varyingenvironments. Warming the substrate 106 provides the benefit ofstimulating mosquitoes during testing. The circulating water heater 108contains a digital control for setting different temperatures and awater tube 124 extending from the heater 108 and running underneath thedisk 122 and the substrate 106 to keep the substrate warm. It is furthercontemplated that any type of heater or heating system may be used towarm substrate 106 to about 35° C. to about 39° C. or any other desiredtemperature or temperature range.

The human surrogate apparatus of FIG.1 is intended for use in alaboratory or controlled environment. Preferably, the controlledenvironment has a temperature range of between about 24° C. to about 30°C. and a range of relative humidity of between about 40% to about 60%.In contrast, FIGS. 2 and 3 illustrate an embodiment of a human surrogateapparatus 200 for testing the efficacy of a skin-applied insectrepellent in the field. By fashioning the human surrogate apparatus 200to be portable, the apparatus may be used in any number of real worldenvironments. The human surrogate apparatus 200 therefore provides theadvantage of similar testing efficacy as human testing, but withoutexposure to mosquitoes or other insects found in the field.

With reference still to FIGS. 2 and 3, the human surrogate apparatus 200is disposed in an outdoor environment, with an identified population ofbiting insects. The portable human surrogate apparatus 200 is providedwith a housing 202 having a power supply line 204 connected thereto. Insome embodiments, a power supply 206 is located remotely from thehousing 202 to provide power to the human surrogate apparatus 200.Alternatively, or in addition, a local power supply is provided withinor attached to the housing 202. In such an embodiment, the power supplyline 204 may be omitted, provided within the housing 202, or otherwisefashioned differently within the housing 202 to allow energy to reachelectrical components thereof.

With reference to FIG. 3, the human surrogate apparatus 200 includes aheater 208 in electrical communication with the power supply 206. In thepresent embodiment, the heater 208 is a silicone-rubber heating blanket,manufactured by BriskHeat Corporation, Columbus, Ohio. A non-collagenbased, non-biological substrate 210 is also provided and is exposed tothe ambient environment. In certain embodiments the non-collagen based,non-biological substrate 210 may be synthetic.

In the present embodiment, the substrate 210 is securely fastened to theportable human surrogate apparatus 200 by magnetic strips 212, or may befastened by any other suitable means. In some embodiments a temperaturebuffering device 214 is provided beneath the substrate 210, whichassists in transmitting heat from the heater 208 and maintains thesubstrate 210 at a predetermined temperature. In the present embodiment,the temperature buffering device 214 is a water bath, enclosed in acontainer 220, which is made of materials that prevent water leakage andthat transmit heat at the same time. Moreover, the portable humansurrogate apparatus 200 is connected to a CO₂ source 216, which may beremote from the housing 202, attached to the housing 202, or otherwiseretained within the housing 202. In the present embodiment, the CO₂source 216 delivers CO₂ at a rate of between about 400 mL/min (14oz/min) to about 600 mL/min (20.3 oz/min).

In certain embodiments, the substrate used for both the laboratory andfield testing is a skin substitute substrate. In some embodiments theskin substitute substrate is a non-biological testing substrate withsurface properties similar to those of human skin. In some embodimentsthe skin substitute substrate is synthetic. In some embodiments the skinsubstitute substrate simulates, i.e., exhibits the same or substantiallysimilar properties, of human skin in various aspects. Such simulatedaspects include one or more of the mechanical, optical, thermal,electrical, chemical, or surface properties of human skin, such assurface roughness, surface optical reflection, and surface wettingability. In some embodiments, one or more, or two or more, or three ormore, or four or more, or five or more, or all of these attributes, arefound in the substrates 106, 210. The non-biological, non-collagen-basedsubstrate according to an embodiment of the present disclosure may beused in a multi-cycle testing method, wherein collagen based substratescannot.

Numerous potential substrates exist that can simulate a human testsubject, of which one or more of them may be taken singly or incombination to effect the purposes of the disclosed human surrogateapparatuses 100, 200. For example, various liquid suspensions can beused to simulate scattering and absorption properties of the skin.Particularly, gelatinous substances are used to simulate variousphysical, mechanical, and chemical properties, such as the elasticmodulus, hardness, optical, or surface properties of the skin.Representative gelatinous substances used in the production of skinsubstitutes are gelatine, agar and agarose, collagens, and polyvinylalcohol gels. Gelatine provides a matrix of density, stiffness, soundspeed, absorption, and light scattering similar to that of human skin.

Further, elastomers can be used in making artificial skin substrates.Elastomers are polymers exhibiting rubber-like viscoelastic properties.Elastomers comprise a broad spectrum of natural and synthetic materials,inter alia silicones, polyurethanes, polyether block amides,polyisoprene, and polybutadiene. Specifically, silicone elastomers suchas cross-linked polydimethylsiloxanes are suitable to generate skinsubstitute substrates.

Moreover, epoxy resins, which have a thermal diffusivity of betweenabout 0.070 to about 0.084 mm²/s (1×10⁻⁴-1.3×10⁻⁴ inches²/s), which isclose to that of human skin, make them another suitable choice for skinsubstitute substrates.

In some embodiments the skin substitute substrate is a synthetic(non-biological) testing substrate. In some embodiments, the substrateused for both laboratory and field testing is a synthetic(non-biological) testing substrate. In some embodiments the skinsubstitute substrate has a critical surface tension and ionic forceidentical, or substantially similar to, human skin. The substrate isnon-collagen based, e.g., the substrate is not a collagen membrane. Inone embodiment as illustrated herein, the skin substitute is aVitro-Skin® substrate (manufactured by IMS, Inc., Portland, Me.),

In certain embodiments, the skin substitute substrate comprises thefollowing characteristics. For example, surface topography in terms ofsurface roughness was evaluated in the skin substitute substrate.Surface roughness, often shortened to roughness, is a component ofsurface texture. It is quantified by the deviations in the direction ofthe normal vector of a real surface from its ideal form. If thesedeviations are large, the surface is considered rough; if they aresmall, the surface is considered smooth. Moreover, roughness plays animportant role in determining how a real object will interact with itsenvironment. R_(a) is the most commonly used roughness parameter. R_(a)is calculated as the Roughness Average of a surface's measuredmicroscopic peaks and valleys. R_(a) is the arithmetic average of theabsolute values of the profile height deviations from the mean line,recorded within the evaluation length. In other words, R_(a) is theaverage of a set of individual measurements of a surface's peaks andvalleys. The reported surface roughness values of human skin on certainbody parts varies in the range R_(a)=0.03 through 0.45 μm. In case ofvery rough surfaces, up to R_(a)=90 μm (Hendrik, C. P. and Franklin, S.E., Influence of Surface Roughness, Material and Climate Conditions onthe Friction of Human Skin. Tribology Letter (2010) Vol. 37, Issue 2, pp361-373.) In certain embodiments the R_(a) value of the skin substitutesubstrate according to the present disclosure was about 0.10±0.07 μm(n=7), which is within the range of surface roughness values of humanskin. R_(a) measurements were made using a Starrett SR300 surfaceroughness meter (manufactured by the L.S. Starrett Company, Athol,Mass.).

Further, the surface topography of the skin substitute substrate wascharacterized by measuring its wavelength dependent optical reflection.The optical reflection was determined using a Shimadzu UV-2600 UV-VisSpectrophotometer (manufactured by Shimadzu Scientific Instruments,Inc., Columbia, Md.) and the results were demonstrated in specularreflectance (FIG. 9A) and diffuse reflectance (FIG. 9B). Specularreflection, also known as regular reflection is the mirror-likereflection of waves, such as light, from a surface. In this process,each incident ray is reflected, with the reflected ray having the sameangle to the surface normal as the incident ray. Diffuse reflection isthe reflection of light from a surface such that an incident ray isreflected at many angles rather than at just one angle as in the case ofspecular reflection. An illuminated ideal diffuse reflecting surfacewill have equal luminance from all directions which lie in thehalf-space adjacent to the surface (Lambertian reflectance). Referringto FIG. 9A, a specular reflectance peak of the skin substitute substratewas identified at a wavelength of 306 nm. Referring to FIG. 9B, thediffuse reflectance exhibited the properties associated with aLambertian reflectance.

Additionally, the surface wetting of insect repellents on the skinsubstitute substrate should be close to the surface wetting of insectrepellents on human skin. The term “wetting” refers to the ease withwhich a substance can intimately contact and spread over a givensubstrate. There are a variety of forces (ionic, static, polar, van derWaals etc.) acting between the substance and the substrates that ensuregood bonding. Good wetting provides a larger area of contact where theseforces may act. Consequently, good wetting is crucial for good bondformation. Insect repellant actives, such as para-menthane-3,8-diol(PMD), DEET, and 2-(2-hydroxyethyl)-1-piperidinecarboxylic acid1-methylpropyl ester (picaridin), were tested on a hydrated skinsubstitute substrate where substantially complete wetting was observed,similar to testing on human skin. Complete wetting is achieved when thecontact angle between the liquid and the solid surface is approaching oris substantially zero. Also, substantially complete wetting demonstratesfavorable interactions between the active substance and the tested skinsubstitute substrate. The interaction of ethanol solutions containing5%, 10%, 15%, and 25% of each aforementioned active substance and thetested skin substitute substrate was also tested. Again, substantiallycomplete wetting was observed for each tested ethanol solution on thetested skin substitute substrate. Due to this degree of wetting betweenthe insect repellent active and the tested skin substitute substrate,the desired bonding between the active and the tested skin substitutesubstrate is achieved. As a result, the insect repellent remains on theskin substitute substrate for a duration similar to that of human skin.

With reference to FIG. 4, a method of using the human surrogateapparatus 100 is explained. Further, data from using this method willprovide experimental evidence of the efficacy of the use of the humansurrogate apparatus 100 in view of other conventional techniques. Atstep 300, approximately 200 adult female mosquitoes are extracted froman insectary cage and are transferred to the container 102. In step 302,the container 102 is placed in the testing room under a particularcondition for about 30 minutes to acclimate the mosquitoes to thepredetermined testing condition. For example, the predetermined testingcondition of the present embodiment has a temperature range of betweenabout 24° C. to about 30° C. and a range of relative humidity of betweenabout 40% to about 60%. Further, in step 304, the heater 108 is turnedon and the temperature set to between about 24° C. to about 30° C.,warming substrate 106. In the present embodiment, substrate 106comprises Vitro-Skin®. The surface temperature of substrate 106 isfurther confirmed at step 306 to be at the predetermined level, e.g., bya laser thermometer or any other thermometer that is capable ofmeasuring surface temperature.

Upon the determination that the appropriate temperature of substrate 106has been reached, a test substance, which can be applied on skin,containing insect repellent is applied to substrate 106 at step 308. Incertain embodiments, different test substances contain differentconcentrations of testing insect repellent. The concentration of testinginsect repellent can be measured by weight percentage or by volumepercentage of the testing insect repellent diluted in ethanol or in anyother suitable diluents. In the present embodiment, about 0.19 g ofskin-applied test substance, i.e., the combined mass of the repellentand diluent, is applied to the warmed substrate during step 308.Further, in the present embodiment, regardless of the concentration ofthe repellent, the combined mass of the repellent and ethanol is about0.19 g. In other experiments, the combined mass of the repellent(s) andthe diluent(s) is greater or less than about 0.19 g, but similarly staysthe same independent of the concentration of the repellent. Withdifferent sets of experiments, the amount of skin-applied test substanceused in the testing is based on the size of the substrate and theproperties of different testing insect repellents, such as the activeingredient dosage. For example, the greater the surface area of thesubstrate, the larger the amount of test substance that is used duringthe testing.

Moreover, during step 310, another similarly warmed substrate 106 istreated with ethanol without any insect repellent. In step 312, thecontainer 102 and the substrate from step 310 are placed incommunication with one another and the mosquitoes are exposed tosubstrate 106. The number of mosquitoes that land on substrate 106 fromstep 310 for equal to or greater than about 2 seconds (i.e., a “bite”)is recorded in step 314. Next, a query is undertaken at step 316. If thenumber recorded in step 314 is smaller than 20 in about 60 seconds, themosquitoes will need to be replaced with new mosquitoes from theinsectary cage at step 300. If the number recorded in step 314 is notless than 20 in about 60 seconds, the testing method will proceed tostep 318, which requires removal of substrate 106 of step 310 fromcommunication with container 102 and placing substrate 106 from step 308into communication with the container 102 and exposing mosquitoes tosubstrate 106 for about 60 seconds.

During Step 318, the treated skin substitute substrate 106 is exposed tobiting insects for a first time period of about X minutes, withdrawnfrom the biting insects for a second time period of about Y minutes, andsubsequently exposed for the first time period of about X minutes to thebiting insects again. This cycle of exposure-withdrawal-exposure isrepeated until one or more biting insects land on the treated substratefor equal to or longer than 2 seconds. In an alternative embodiment,during step 318, the treated substrate is exposed to biting insectswithin a range of about 1 to about 5 minutes and withdrawn for a rangeof about 25 to about 29 minutes. Step 318 is repeated for n times (n≥1)until one or more mosquitoes land on the treated substrate 106 duringthe (n)th exposure and during the subsequent (n+1)th exposure. At step320, exposure of mosquitoes to the treated substrate 106 is terminatedand the time needed to obtain the first bite during the (n)th exposureis recorded as the CPT for measuring the efficacy of the skin-appliedinsect repellent. Collagen-based substrates do not hold up and cannot beused in such a multi-cycle testing protocol.

When comparing the CPT data of skin-applied insect repellents, such asDEET, using the human surrogate apparatus 100 to the CPT data ofskin-applied insect repellents obtained using human researchparticipants via the arm-in-cage method (See Product Performance TestGuidelines OPPTS 810.3700: Insect Repellents to be Applied to HumanSkin, United States Environmental Protection Agency, EPA 712-10-001,Jul. 7, 2010), data from both groups is very similar to each other asmay be seen with reference to FIG. 5 in a controlled environment, e.g.,a laboratory. Further, Table 1 illustrates the average CPT for the twomethods based on differing weight percentages of the active ingredientDEET of the insect repellent, as well as the standard deviation (“±SE”)at each weight percentage for the two groups. As may be seen, thedifferences between the experiments using human research participantsvia the arm-in-cage method and the experiments using the human surrogateapparatus 100 are statistically negligible.

TABLE 1 Mean (±SE) and median CPT in hours for different concentrationsof DEET in ethanol (EtOH) using the arm in cage method and humansurrogate apparatus 100 method. Complete protection time Mean ± SE(median) Concentration Human Skin (% DEET) Arm in cage Surrogate 7 2.5 ±0.7 (2.3) 2.2 ± 0.9 (2.0) 15 3.9 ± 1.5 (3.5) 3.6 ± 1.0 (3.3) 20 4.9 ±2.1 (3.8) 4.9 ± 2.1 (5.3) 25 6.5 ± 1.4 (6.8) 6.3 ± 1.1 (6.5) 30 6.6 ±0.7 (6.6) 6.5 ± 2.3 (7.0)

With reference to FIG. 6, the mean CPT (±SE) between the two groups fortesting different weight percentages of DEET in ethanol is displayed.Similarly, there is a statistically negligible difference between theexperiments using human research participants via the arm-in-cage methodand the experiments using the human surrogate apparatus 100.

Further, with reference to FIG. 7, comparison of five differentnon-linear, sigmoid models for the arm-in-cage method and the humansurrogate apparatus 100 method have been performed and the best fitmathematical model for CPT is selected. Particularly, the model with thelowest corrected Akaike information criterion (AICc) and Bayesianinformation criterion (BIC) is selected as the model with the best fitto the data. Curve 400 of the arm-in-cage group demonstrates the CPTincreases as the DEET concentration increases, and curve 402 of thehuman surrogate apparatus 100 group demonstrates a very similarcorrelation.

Moreover, when inversely predicting the average (±SE) weight percentageof DEET that corresponds to a given CPT in hours, the differences of thepredicted weight percentages of DEET between the arm-in-cage group andthe human surrogate apparatus 100 are minimal and statisticallynegligible, as may be seen in Table 2.

TABLE 2 Estimates are calculated for 1-6 h of complete protection time.Predicted percent DEET Complete concentration (±SE) protection timeHuman Skin (h) Arm in cage Surrogate 1  1.61 ± 1.69  3.81 ± 1.72 2  6.15± 0.94  8.17 ± 1.16 3 10.07 ± 0.88 11.92 ± 1.04 4 14.08 ± 1.15 15.71 ±1.13 5 18.70 ± 1.46 20.00 ± 1.21 6 24.85 ± 2.64 25.55 ± 1.58

Additionally, when other insect repellents are tested, such as picaridinand PMD, the CPT data obtained in the group using human surrogateapparatus 100 shares overlapping confidence intervals with, and is notsignificantly different from, the data obtained in the group using thearm-in-cage method in a laboratory. For example, referring to FIG. 14,the CPT data points at 10% by weight of picaridin of the group usinghuman surrogate apparatus 100 are within the range of CPT data points ofthe group employing the arm-in-cage method. Similarly, referring to FIG.15, the CPT data at 10% by weight of PMD of the human surrogateapparatus 100 group is not significantly different from the CPT data ofthe arm-in-cage group.

It has also been experimentally determined that the human surrogateapparatuses described herein perform consistently in a controlledenvironment, e.g., a laboratory with predetermined and controllableconditions, and in an uncontrolled environment, e.g., in an outdoorfield test. With reference to FIG. 8 and Table 3, the CPT in hoursobtained from testing 7% weight percentage DEET repellent efficacy inthe laboratory and obtained from the field is similar. Therefore,utilization of either of the human surrogate apparatuses 100, 200 doesnot affect testing results.

TABLE 3 Mean (±SE), median, and range of CPT in hours conducted in thelaboratory and field using 7% DEET in EtOH. Complete protection time (h)Setting Mean ± SE Median Range Field 2.0 ± 0.4 2.0 1.5-2.5 Lab 2.2 ± 0.92.0 1.0-3.5

The CPT data of skin-applied insect repellents using human surrogateapparatus 200 and the CPT data of skin-applied insect repellentsobtained using voluntary human research participants outdoor (pleaserefer to OPPTS 810.3700. Insect repellents for human skin and outdoorpremises athttps://rchive.epa.gov/scipoly/sap/meetings/web/pdf/insectguid.pdf foroutdoor field test protocols) are comparable. For example, referring toFIGS. 10A-10C, the CPT data (including average CPT) of DEET in the humansurrogate apparatus 200 group shares overlapping confidence intervalswith the CPT data in the field test group using voluntary humansubjects, i.e., the CPT data in the human surrogate apparatus 200 groupis not significantly different from the CPT data in the voluntary humansubjects group. Further, referring to FIG. 10B, curve 500 of thevoluntary human subjects group demonstrates the CPT increases as theDEET concentration increases in the field and curve 502 of the humansurrogate apparatus 200 group demonstrates a very similar correlation.Similarly, for testing substance picaridin (referring to FIGS. 11A-11C)and PMD (referring to FIGS. 12A-12C), the CPT data (including averageCPT) in the human surrogate apparatus 200 group shares overlappingconfidence intervals with the CPT data in the field test group using thevoluntary human subjects. No statistically significant difference isobserved in either the picaridin outdoor field test or the PMD outdoorfield test when comparing groups involving human participants to groupsinvolving the human surrogate apparatuses.

The human surrogate apparatus can also be used in evaluating mosquitolanding pressure (also known as landing frequency), which representsmosquitoes' intent to bite according to the EPA. In certain embodiments,human surrogate apparatus 200 is utilized in recording the number ofwild mosquitoes that land on the human surrogate apparatus during aperiod of time. Referring to FIG. 13 and Table 4, the number ofmosquitoes reported for the human surrogate apparatus, which is thenumber of mosquitoes that landed on the apparatus over a 1-minuteperiod, was significantly higher than the numbers of mosquitoes capturedby the industry standard mosquito traps, such as the CDC Miniature LightTrap(http://johnwhock.com/products/mosquito-sandfly-traps/cdc-miniature-light-trap/)and the BG Sentinel-2 trap (Biogents,http://www.biogents.com/bg-sentinel/) over a one hour period. In FIG.13, “total all spp” is the total number of mosquitoes of all speciescaptured by the CDC or BG trap or that landed on the human surrogateapparatus. Due to the human surrogate apparatus' ability to recordlandings, a capture step is not necessary, making the human surrogateapparatus a better indicator of mosquito activity in a given area.Gathering mosquito activity in an area is very important in determiningpotential for disease transmission, etc.

TABLE 4 Summary statistics Mean number of Device mosquitoes Non-humandevice 141.7* CDC Miniature Light Trap 33.8** BG Sentinel-2 28.7***Number of mosquito lands over a 1-minute period **Number of mosquitoescaptured over a 1-hour periodIndustry-standard mosquito traps are not ideal for evaluating mosquitoeslanding pressure in an outdoor setting because mosquitoes have to passthrough certain barriers of the traps, whereas, using voluntary humansubjects in a similar evaluation would not require mosquitoes to passthrough any barriers. However, as aforementioned, there are severaldisadvantages associated with using voluntary human subjects. Based onthe results demonstrated in FIG. 13 and table 4, the human surrogateapparatus disclosed in the current application is ideal to replacevoluntary human subjects in evaluating mosquitoes landing pressure andbiting pressure (also known as biting frequency).

Any of the embodiments described herein may be modified to include anyof the structures or methodologies disclosed in connection withdifferent embodiments.

INDUSTRIAL APPLICABILITY

A human surrogate apparatus for testing the efficacy of a skin-appliedinsect repellent is presented that is comparable in results to testinginvolving human participants.

Numerous modifications to the present invention will be apparent tothose skilled in the art in view of the foregoing description.Accordingly, this description is to be construed as illustrative onlyand is presented for the purpose of enabling those skilled in the art tomake and use the invention and to teach the best mode of carrying outsame. The exclusive rights to all modifications which come within thescope of the appended claims are reserved.

I/We claim:
 1. An apparatus, comprising: a housing comprising anaperture extending through a wall of the housing; a carbon dioxidedelivery device coupled to the housing through the aperture; anon-biological skin substitute substrate; and a heater coupled to thenon-biological skin substitute substrate.
 2. The apparatus of claim 1,wherein the non-biological skin substitute substrate simulates at leastone physical, chemical, or biological characteristic of human skin. 3.The apparatus of claim 2, wherein the non-biological skin substrate issynthetic.
 4. The apparatus of claim 2, wherein the non-biological skinsubstitute substrate has an R_(a) value of about 0.01 to about 0.2 μm.5. The apparatus of claim 2, wherein the non-biological skin substitutesubstrate has specular reflectance peak at a wavelength of about 306 nm.6. The apparatus of claim 2, wherein the non-biological skin substitutesubstrate has a diffuse reflectance exhibiting properties associatedwith a Lambertian reflectance.
 7. The apparatus of claim 2, wherein thecontact angle between an insect repellent and a surface of thenon-biological skin substitute substrate is substantially zero when theinsect repellent is applied to the non-biological skin substitutesubstrate.
 8. An apparatus, comprising: a portable housing with acavity; a heater disposed within the cavity of the housing; atemperature buffering device coupled to the heater and disposed withinthe cavity of the housing; a non-biological skin substitute substratecoupled to the temperature buffering device and disposed within thecavity of the housing; and a carbon dioxide delivery device coupled tothe housing.
 9. The apparatus of claim 8, wherein the non-biologicalskin substitute substrate simulates at least one physical, chemical, orbiological characteristic of human skin.
 10. The apparatus of claim 9,wherein the non-biological skin substrate is synthetic.
 11. Theapparatus of claim 9, wherein the non-biological skin substitutesubstrate has an R_(a) value of about 0.01 to about 0.2 μm.
 12. Theapparatus of claim 9, wherein the non-biological skin substitutesubstrate has specular reflectance peak at a wavelength of about 306 nm.13. The apparatus of claim 9, wherein the non-biological skin substitutesubstrate has a diffuse reflectance exhibiting properties associatedwith a Lambertian reflectance.
 14. A method for measuring mosquitolanding pressure, comprising the steps of: providing a non-biologicalskin substitute substrate; exposing a plurality of mosquitoes to theskin substitute substrate; and recording a number of mosquitoes landingon the skin substitute substrate during a period of time.
 15. The methodof claim 14, wherein the period of time is about 1 minute.
 16. Themethod of claim 14, wherein the non-biological skin substitute substratesimulates at least one physical, chemical, or biological characteristicof human skin.
 17. The method of claim 14, wherein the non-biologicalskin substrate is synthetic.
 18. The method of claim 14, wherein thenon-biological skin substitute substrate has an R_(a) value of about0.01 to about 0.2 μm.
 19. The method of claim 14, wherein thenon-biological skin substitute substrate has a specular reflectance peakat a wavelength of about 306 nm.
 20. The method of claim 14, wherein thenon-biological skin substitute substrate has a diffuse reflectanceexhibiting properties associated with a Lambertian reflectance.